Fertilizers containing microorganisms and their production processes

ABSTRACT

Fertilizer containing microorganisms of the rhizosphere packaged by microencapsulation in a polysaccharide matrix. The inoculum obtained after microencapsulation can be mixed with a granular solid fertilizer, then stored, or with a liquid fertilizer immediately before spreading.

BACKGROUND OF THE INVENTION

This invention relates to fertilizers containing microorganisms andprocess for producing such fertilizers.

It is known that plants cultivated on a large scale maintain amicroflora consisting, for example, of bacteria for fixing nitrogenassociated with their roots and that these rhizospheric microflora havean effect of promoting plant growth.

By adding an inoculum of a bacteria of the rhizosphere, a modificationof the root development is observed (elongation, dry weight, degree ofbranching, number of absorbent hairs), a modification of the total drymatter and an increase in the grain yield. These effects are probablydue to a combination of several phenomena such as nitrogen fixation,production of plant growth hormones, and pectinolytic activities thatmake better mineral absorption possible. The influence of the additionof bacteria inocula in the rhizosphere of plants is described, forexample, in the article by Gaskins, M. H., Albrecht, S. L. and Hubbel,D. H. Agric. Ecos. Environ. 1984 (12), pp 99-116.

The methods used by the researchers: inoculation in liquid or granularform independently of any other agricultural practice is hard to achievein the field because of the extra work it would impose on farmers. Ithas been proposed for cultivation of leguminous plants to package someinocula on some peat and to coat the seeds with the product obtained,but this process has the drawback of putting the microorganisms indirect contact with the different pesticides that generally coat grains.Now, these pesticides most often exhibit a high toxicity with respect tomost bacteria species.

It would be advantageous to be able to mix fertilizers and the inoculaof rhizosphere bacteria because it has been shown that the effect of aninoculation was better in the presence of mineral, and in particularnitrogenous, fertilizers. Actually, plants having good mineral nutritionexhibit a great deal of root exudation, which promotes the developmentof rhizospheric microflora. However, up to now the difficulty has beenencountered that the inocula did not have sufficient viability uponcontact with the fertilizer.

BRIEF DESCRIPTION OF FIGURE

FIG. 1 illustrates the survival of Azospirillum left in contact with amedium containing a nitrogeneous solution of SOLONIA S390.

SUMMARY OF THE INVENTION

According to this invention, a form of inoculum has been found that hassufficient viability, upon contact with the fertilizer, for direct usein the field. More specifically, this invention relates to a fertilizercontaining microorganisms isolated from the rhizosphere packaged bymicroencapsulation in a polysaccharide.

Processes of microencapsulating microorganisms in a polysaccharidematrix are already known, in particular from patents EP-A-017,565 andEP-A-083,267. Another usable process according to this inventionconsists in:

preparing a culture of microorganism in a fermenter in the presence ofoxygen,

stopping the oxygen supply and adding, preferably with agitation,polysaccharide to the culture medium,

applying, on the surface of the mixture, an excess pressure of about 0.2to 1.9 bars, depending on the type and concentration of polysaccharideadded,

introducing the mixture, under pressure, into a device comprising atleast one calibrated nozzle with a diameter of about 0.1 to 0.8 mm,

spraying the mixture, with the nozzle, in the form of drops into amineral salt solution,

rinsing and drying the gel obtained to obtain a storable inoculum.

The mineral salt used in this process can be, in particular, a salt suchas chloride or sulfate of iron, manganese, aluminum, calcium, zinc orcopper.

The encapsulated microorganism is generally in the form of a light beigepowder able to be mixed with any fertilizer containing nitrogen and/orphosphorus and/or potassium. It more advantageously mixed withnitrogenous fertilizers such as ammonium nitrate or urea.

The fertilizer can be in solid form, in suspension or in liquid form. Inthe case of a solid fertilizer, the granular fertilizer can, forexample, be mixed with the powder of the microencapsulated inoculum in adrum until the mixture is homogeneous. The respective quantities offertilizer and microencapsulated inoculum are generally such that theweight ratio of the fertilizer to the microencapsulated inoculum isbetween about 500 and 2,000. Then a coating product such as an amine, anatural wax or a synthetic wax can be sprayed on the granules offertilizer.

The coating product notably has the advantage that, when used in thisinvention, it efficiently achieves a homogeneous adhesion of theinoculum. The fertilizer according to the invention can then be stored.

In the case where the fertilizer is in suspension or solution, thefertilizer and inoculum are mixed immediately before application, thenthe entire unit is applied on the field to be fertilized.

The examples given below in a nonlimiting way will make it possible tobetter understand the invention.

EXAMPLE 1

Azospirillum lipoferum, a microorganism isolated from the rhizosphere ofcorn, is cultured in a medium containing, per liter:

    ______________________________________                                        MgSO.sub.4, 7H.sub.2 O                                                                              200    mg                                               NaCl                  100    mg                                               CaCl.sub.2            20     mg                                               FeSO.sub.4, 7H.sub.2 O                                                                              10     mg                                               Na.sub.2 MoO.sub.4, 2H.sub.2 O                                                                      2      mg                                               yeast extract         3      mg                                               KH.sub.2 O.sub.4      400    mg                                               K.sub.2 HPO.sub.4     600    mg                                               glucose               30     g                                                NH.sub.4 Cl           5      g                                                H.sub.2 O             qsp    liter                                            ______________________________________                                    

The air throughput and the agitation speed are such that the dissolvedoxygen concentration is 30%. The starting pH being 6.8, it develops to7.1 then is kept at this value by adding 1N soda.

The microencapsulation of this inoculum is performed with a solution ofsodium alginate at the rate of 10 g/l. The mixture is stirred at a speedof 600 rpm. The solution is subjected to a pressure of 0.5 bars, then itis dispersed in a CaCl₂ solution at 6 g/l.

The gel obtained is rinsed with water, then it is oven-dried at atemperature of 30° C. with a relative humidity of 30%.

EXAMPLE 2

A quantity A of the microencapsulated inoculum of example 1 is mixed forabout 5 minutes with a quantity B of granular ammonium nitrate in agranulation drum. The weight ratio B/A is 1,000. A quantity C of anaminated oil sold under the name LILAMINE AC 81L is sprayed on thegranules thus obtained. In solidifying, this amine comes to coat theammonium nitrate granules and a homogeneous adhesiveness of the inoculumis obtained. The weight ratio of A/C is 1.

Then the number of viable bacteria is measured according to thefollowing protocol:

2 g of ammonium nitrate is dissolved in 500 ml of sterile distilledwater. The powdered inoculum is collected by filtration on a filter witha 0.45-micrometer porosity.

the filter is placed in 50 ml of phosphate buffer having the followingcomposition:

    ______________________________________                                        KH.sub.2 PO.sub.4     3.336 g                                                 Na.sub.2 HPO.sub.4   18.773 g                                                 H.sub.2 O            qsp 1 liter                                              ______________________________________                                    

and having a pH of 7.2. The polysaccharide is decrosslinked in a buffersolution.

This operation is facilitated by putting the flask used on an orbitalagitating table for 1 hour. Decimal dilutions are spread on a Petri dishand the colonies that have appeared after incubation for 48 to 72 hoursat a 35° C. temperature, the optimal temperature for growth ofAzospirillum lipoferum, are counted.

The following viability is observed:

1.6×10⁹ viable cells after 26 hours of storage.

1.5×10⁹ viable cells after 3 days of storage.

0.7×10⁹ viable cells after 9 days of storage.

0.3×10⁹ viable cells after 1 month of storage.

EXAMPLE 3

The microencapsulated inoculum of the example is left in contact in anitrogenous solution sold under the name SOLONIA S 390 having thefollowing composition by weight:

    ______________________________________                                               CO(NH.sub.2).sub.2                                                                    33.2%                                                                 NH.sub.4 NO.sub.3                                                                     41.5%                                                                 H.sub.2 O                                                                             25.3%                                                          ______________________________________                                    

The solution containing the bacteria is filtered as is and at regularintervals the residual viability of the bacteria is measured accordingto the protocol of example 2. The accompanying curve illustrates thesurvival of bacteria in such a medium.

It is interesting to note that most of the liquid spreaders empty inabout 0.5 hours. As a result, the inoculum can be spread while itexhibits a high viability compatible with the doses used duringbacterial inoculation in the field.

We claim:
 1. A mixture of fertilizer and a minor amount ofmicroorganisms isolated from the rhizosphere, said microorganisms beingpackaged by microencapsulation in a polysaccharide matrix.
 2. Afertilizer mixture according to claim 1, in which the microencapsulationis performed according to a process comprising:preparing a culture ofmicroorganism in a fermenter in the presence of oxygen, stopping theoxygen supply and adding polysaccharide to the culture medium, applying,on the surface of the mixture, an excess pressure of 0.2 to 1.9 bars,introducing the mixture under pressure into a device comprising at leasta calibrated nozzle with a diameter of 0.1 to 0.8 mm, spraying themixture, with the nozzle, in the form of drops into a mineral saltsolution, rinsing and drying the gel obtained to obtain a storableinoculum.
 3. A fertilizer according to claim 1, characterized in that itis in solid form.
 4. A fertilizer mixture according to claim 3, whereinthe fertilizer is a nitrogenous fertilizer.
 5. A fertilizer mixtureaccording to claim 4, wherein the weight ratio of the fertilizer to themicroencapsulated microorganism is between about 500 and
 2000. 6. Afertilizer mixture according to claim 3, wherein the fertilizer isgranular, said mixture being provided with a coating resulting in aproduct having a homogeneous adherence of the microencapsulatedmicroorganism on the granular fertilizer.
 7. A fertilizer mixtureaccording to claim 6, wherein the coating is an amine or wax.
 8. Afertilizer mixture according to claim 6, wherein the coating is anaminated oil.
 9. A fertilizer mixture according to claim 8, wherein themixture is homogenous, the fertilizer is nitrogenous, and the weightratio of the fertilizer to the microencapsulated microorganism isbetween about 500 and
 2000. 10. A fertilizer mixture according to claim9, wherein the microorganism comprises Azospirillum lipoferum.
 11. Afertilizer mixture according to claim 1, wherein the fertilizer is inliquid form.
 12. A fertilizer mixture according to claim 11, prepared bymixing said fertilizer and the microencapsulated microorganismsimmediately before spreading.
 13. A fertilizer mixture according toclaim 12, wherein the fertilizer is a nitrogenous fertilizer.
 14. Afertilizer mixture according to claim 13, wherein the weight ratio ofthe fertilizer to the microencapsulated microorganism is between about500 and
 2000. 15. A fertilizer mixture according to claim 13, whereinthe microorganism comprises Azospirillum lipoferum.
 16. A fertilizermixture according to claim 1, wherein the fertilizer is a nitrogenousfertilizer.
 17. A fertilizer mixture according to claim 16, wherein theweight ratio of the fertilizer to the microencapsulated microorganism isbetween about 500 and
 2000. 18. A fertilizer mixture according to claim16, wherein the microorganism comprises Azospirillum lipoferum.
 19. Afertilizer mixture according to claim 1, wherein the weight ratio of thefertilizer to the microencapsulated microorganism is between about 500and
 2000. 20. A fertilizer mixture according to claim 1, wherein themicroorganism comprises Azospirillum lipoferum.